This invention relates to vaccines, and more particularly to vaccine adjuvants.
Immune responses to many different antigens (e.g., microbial antigens or tumor antigens), while detectable, are frequently or insufficient magnitude to afford protection against a disease process mediated by agents (e.g., infectious microorganisms or tumor cells) expressing those antigens. In such situations, it is often desirable to administer to an appropriate subject, together with the antigen, an adjuvant that serves to enhance the immune response to the antigen in the subject.
The invention derives from the finding that fusion agents (e.g., fusion proteins) containing an immune enhancing domain and an immunogenic domain induced more potent Th1 type T cell responses than did the molecule contained in the immunogenic domain when administered alone to mice. A similar pattern of immune responsiveness was observed when a naked DNA construct encoding a fusion protein was administered to the mice. Thus, the invention features fusion agents containing an immune enhancing domain and an immunogenic domain. In preferred embodiments, both domains are proteins and, where they are, the relevant fusion agents are called xe2x80x9cfusion proteins.xe2x80x9d The invention also features DNAs encoding fusion proteins, vectors containing the DNAs, cells containing the vectors, and methods of making and using the fusion agents and DNAs.
More specifically, the invention features a fusion agent comprising a first domain and a second domain. The first domain contains an immune enhancing molecule and the second domain comprises an immunogenic molecule. The immunogenic molecule can be a tumor antigen, an autoantigen, a molecule produced by a fungus, a molecule produced by a mycoplasma, a molecule produced by a yeast, a polypeptide encoded by a virus, or a molecule produced by a bacterium. The bacterium can be Salmonella enteriditis, Listeria monocytogenes, Mycobacteria leprae, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Borrelia burgdorferi, Actinobacillus pleuropneumoniae, Helicobacter pylori, Neisseria meningitidis, Yersinia enterocolitica, Bordetella pertussis, Porphyromonas gigivalis, or a mycoplasma (e.g., Mycoplasma hyopneumoniae). The immune enhancing molecule is a protein or a functional fragment of the protein, e.g., Mycobacterium tuberculosis Early Secretory Antigenic Target 6 (ESAT-6). The immunogenic molecule can be a protein or a functional fragment of the protein, e.g., Mycoplasma hyopneumoniae P71 protein. In addition, the fusion agent can be a fusion protein. The fusion agent can contain one or more additional domains, each of which can contain an immune enhancing molecule or an immunogenic molecule.
The invention also encompasses a DNA encoding a fusion protein that includes an immune enhancing domain and an immunogenic domain, each with the above described characteristics. In addition, the invention includes a vector containing the DNA of the invention. The vector can contain a transcriptional regulatory element (TRE) operably linked to the DNA. Also embraced by the invention is a cell containing any of the vectors of the invention.
The invention features a method of making a fusion protein. The method involves: (a) culturing a cell of the invention in which the vector that the cell contains includes a TRE operably linked to the DNA; and (b) extracting the fusion protein from the culture.
In another aspect, the invention encompasses a method of inducing an in vitro immune response to an immunogenic molecule, the method comprising culturing the a fusion agent of the invention with a T cell and an antigen presenting cell (APC). The invention also features a method of inducing an in vivo immune response to an immunogenic molecule. This method involves delivering the fusion agent (e.g., a fusion protein of the invention) to an immune system of a subject. The delivery can involve administering the fusion agent to the subject. Alternatively, where the fusion agent is a fusion protein, the delivery can involve administering a vector of the invention to the subject. The vector can include (a) a DNA encoding the fusion protein; and (b) a TRE operably linked to the DNA. The immune response to the immunogenic molecule can be an interferon-xcex3 producing immune response and/or an IgG2a antibody response.
Another embodiment of the invention is a DNA that includes a nucleotide sequence encoding: (a) a Mycoplasma hyopneumoniae P71 protein; or (b) a functional fragment of the P71 protein. In this DNA, codons encoding tryptophan in the protein or the functional fragment are not TGA codons.
xe2x80x9cPolypeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.
As used herein, an xe2x80x9cimmune enhancing moleculexe2x80x9d is a molecule (e.g., a polypeptide) that, when administered to a mammalian subject in the form of a fusion agent also containing an immunogenic molecule, elicits a more potent IFN-xcex3-producing response to the immunogenic molecule than would the immunogenic molecule administered alone to the mammalian subject. It is understood that the immune enhancing molecule, when administered to the subject in the form of the fusion agent, can also elicit an IFN-xcex3-producing response to itself. It is also understood that the invention is not limited by any particular mechanism of action. Thus, the cells activated to produce IFN-xcex3 by the fusion agents can be, for example, CD4+ T cells, CD8+ T cells, macrophages, monocytes, or any other cell capable of producing IFN-xcex3.
As used herein, an xe2x80x9cimmunogenic moleculexe2x80x9d is a molecule that can elicit an immune response when administered to a mammalian subject on its own and/or together with an immune enhancing molecule in the form of a fusion agent. Thus, an immunogenic molecule, as used herein, is not necessarily capable of eliciting an immune response in a mammalian subject when administered on its own to the mammalian subject.
As used herein, a xe2x80x9cfunctional fragmentxe2x80x9d of an immune enhancing polypeptide that is part of a fusion agent of the invention that also contains an immunogenic molecule is a fragment of the immune enhancing polypeptide that is shorter than the full-length immune enhancing polypeptide and has at least about 10% (e.g., about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% or even more) of the ability of the immune enhancing polypeptide to facilitate a more potent IFN-xcex3-producing response to the immunogenic molecule than the immunogenic molecule would on its own. Methods of establishing whether a fragment of an immune enhancing polypeptide is functional are known in the art. For example, fragments of interest can be made by either recombinant, synthetic, or proteolytic digestive methods. Such fragments can then be isolated and tested for their ability to enhance a IFN-xcex3-producing type response by procedures described herein.
As used herein, a xe2x80x9cfunctional fragmentxe2x80x9d of an immunogenic polypeptide that is part of a fusion agent of the invention that also contains an immune enhancing molecule is a fragment of the immunogenic polypeptide that is shorter than the full-length immunogenic polypeptide and has at least about 10% (e.g., about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% or even more) of the ability of the immunogenic polypeptide to stimulate an immune response to the immunogenic polypeptide. Methods of establishing whether a fragment of an immunogenic polypeptide is functional are known in the art. For example, fragments of interest can be made by either recombinant, synthetic, or proteolytic digestive methods. Such fragments can then be isolated and tested for their ability to stimulate an immune response to itself. Such testing can be performed using the fragment on its own or in the form of a fusion agent together with an immune enhancing molecule of interest. Naturally it will be necessary to do the testing in the form of a fusion agent when comparing the activities of an immunogenic polypeptide and a fragment of such a polypeptide, neither of which alone stimulate an immune response.
As used herein, xe2x80x9coperably linkedxe2x80x9d means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.